PROJECT SUMMARY: Simultaneous optical monitoring of the electrical activity of hundreds of neurons with single-cell fidelity comparable to whole-cell patch clamp electrophysiology would enable recording of the circuit-level activity that gives rise to affect, behavior, and cognition and would drive novel insights into the network dysregulation underlying psychiatric and neurological disorders. Therefore, enormous effort has been expended on designing genetically encoded voltage indicators (GEVIs), cell-type specific protein reporters that transduce changes in membrane potential as a fluorescent signal. Large-scale adoption of GEVIs is dependent on them possessing the brightness, voltage-sensitivity, and temporal resolution to allow for the in vivo recording of high frequency bursts, the monitoring of sub-threshold voltage dynamics, and the accurate reconstruction of action potential waveforms. Current GEVIs, such as those constructed from archaerhodopsin, do not possess all these desired properties and are intrinsically limited in their temporal resolution due to their reliance on structural rearrangements for signal transduction. We propose the construction of high temporal resolution GEVIs by leveraging validated methods in computational de novo protein design to produce transmembrane helical bundle proteins that bind a near-infrared fluorescent, mammalian-endogenous biliverdin chromophore. Proper positioning of biliverdin within the protein will allow for optical voltage-reporting by the Stark effect, an intrinsically voltage-sensitive phenomenon wherein an electric field acting upon a chromophore causes sub- picosecond changes in fluorescence by altering absorbance. This work will produce near-infrared fluorescent, high temporal resolution voltage probes permitting new insights into the circuit-level physiology underlying the complex phenotypes seen in the normal and diseased brain.